SACKLER SPECIAL FEATURE: PERSPECTIVE

Architecture of human telomerase RNA

Qi Zhang, Nak-Kyoon Kim, and Juli Feigon1
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569
Edited by Neal F. Lue, Weill Cornell Medical College, New York, NY, and accepted by the Editorial Board July 22, 2011 (received for review June 8, 2011)

Telomerase is a unique reverse transcriptase that catalyzes the addition of telomere DNA repeats onto the 3′ ends of linear chromosomes
and plays a critical role in maintaining genome stability. Unlike other reverse transcriptases, telomerase is unique in that it is a ribonu-
cleoprotein complex, where the RNA component [telomerase RNA (TR)] not only provides the template for the synthesis of telomere DNA
repeats but also plays essential roles in catalysis, accumulation, TR 3′-end processing, localization, and holoenzyme assembly. Biochemical
studies have identified TR elements essential for catalysis that share remarkably conserved secondary structures across different species as
well as species-specific domains for other functions, paving the way for high-resolution structure determination of TRs. Over the past
decade, structures of key elements from the core, conserved regions 4 and 5, and small Cajal body specific RNA domains of human TR have
emerged, providing significant insights into the roles of these RNA elements in telomerase function. Structures of all helical elements of
the core domain have been recently reported, providing the basis for a high-resolution model of the complete core domain. We review
this progress to determine the overall architecture of human telomerase RNA.

Box H/ACA RNA | NMR | pseudoknot | telomerase reverse transcriptase

T
elomerase is a large, multisubunit
ribonucleoprotein (RNP) that
replicates the 3′ end of linear
chromosomes by processive syn-
thesis of telomere DNA repeats. Telo-
meres, the physical ends of linear
chromosomes, generally comprise dsDNA
with a short repeating species-specific se-
quence ending in a 3′ single-stranded
overhang of variable length plus associated
telomere binding proteins, called shelterin
in humans (1, 2). Telomeres protect the
integrity of linear chromosomes by allow-
ing the cellular DNA repair machinery
to distinguish them from double-strand
breaks, thus playing critical roles in main-
taining genome stability in eukaryotes (1,
2). Shortening of telomeres below a criti-
cal length because of inherent incomplete
replication of DNA ends ultimately leads
to telomere fusions and cell senescence
(3–6). The 3′ ends of telomeres are repli-
cated by telomerase, a unique reverse
transcriptase discovered almost three
decades ago (7), which catalyzes the ad-
dition of telomere DNA repeats onto the
ends of linear chromosomes using an
embedded RNA as the template (8, 9).
Although telomerase has a low or
undetectable level of activity in most so-
matic cells, it is active in some germline,
epithelial, and hematopoietic cells, and it
is highly active in the majority (∼90%) of
cancer cell lines (10–12). Telomerase de-
ficiency because of mutations in human
telomerase RNA (hTR) has also been
linked to several inherited human dis-
eases, such as dyskeratosis congenita,
aplastic anemia, myelodysplasia, and idio-
pathic pulmonary fibrosis (13–26).
The telomerase holoenzyme includes
a unique reverse transcriptase [telomerase
reverse transcriptase (TERT)], an essential
RNA (TR), and several species-specific
proteins required for proper function in
vivo (27–29). The protein TERT is highly
conserved across different species, and it
usually contains four major functional
domains: the TERT N-terminal domain
(TEN), the TERT RNA binding domain
(TRBD), the reverse transcriptase domain
(RT), and the C-terminal extension (27,
30–32). The TEN domain interacts with
the ss telomere DNA repeats, the TRBD
domain binds multiple sites of TR, and the
RT and C-terminal extension domains
bind the RNA/DNA hybrid and catalyze
the addition of DNA repeats onto the 3′
end (27, 31–34). Although no structures of
TERT with all four domains have yet been
reported for any species, structures of the
TEN and TRBD domains from Tetrahy-
mena thermophila telomerase (33, 35) and
the full-length Tribolium castaneum TERT
that lacks the TEN domain have been re-
ported (36, 37). These high-resolution
structures have significantly advanced our
understanding of how TERT catalyzes the
reverse transcription of telomere DNA
and how TERT could potentially interact
with the ss telomere DNA and the tem-
plate RNA/DNA hybrids. Excellent re-
views can be found elsewhere that describe
the current state of knowledge about the
structure and function of TERT (31, 32).
Although the essential templating
function of TR was discovered more than
20 y ago (38), the TR contains more than
a template. To date, the TR sequences of
28 ciliates, 43 vertebrates, and 25 yeasts
have been determined (39–41). In contrast
to the relatively conserved TERT, TRs
differ greatly not only in sequence but also
in length, ranging from 147 to 205 nt in
ciliates (38, 42–45), from 312 to 559 nt
in vertebrates (46, 47), and from 779 to
>2,030 nt in yeasts (40, 41). Despite sig-
nificant challenges in identifying common
functional TR elements because of such
divergence, phylogenetic and mutational
studies have revealed a conserved sec-
ondary structure found in common among
TRs across different species, which in-
cludes a large loop containing the tem-
plate, a 5′ template boundary element,
a pseudoknot, a loop-closing helix, and
a stem terminus element (STE) (9, 15, 46,
48). The conservation of secondary struc-
tures rather than sequences suggests a role
for these RNA structural motifs in telo-
merase function, and indeed, these regions
of TR are essential for synthesis of telo-
mere repeats (49–55). Other regions of
TR are involved in species-specific roles in
telomerase biogenesis, RNA processing,
localization, and accumulation (56–61).
A conserved secondary structure for
vertebrate TRs was first determined on the
basis of 35 sequences (46), with four pro-
posed conserved structural domains: (i)
the pseudoknot, which includes the tem-
plate, and (ii) the conserved regions 4 and
5 (CR4/CR5), which together comprise
the catalytic core of the TR; (iii) the box
H/ACA; and (iv) the CR7. An updated
secondary structure of hTR (47, 62) is
shown in Fig. 1 with known associated
holoenzyme proteins. All of the secondary
structure elements found in common
among TRs across different species are in
the 5′ region of hTR, where the STE is the
This paper results from the Arthur M. Sackler Colloquium
of the National Academy of Sciences, “Telomerase and
Retrotransposons: Reverse Transcriptases That Shaped Ge-
nomes” held September 29–30, 2010, at the Arnold and
Mabel Beckman Center of the National Academies of Sci-
ences and Engineering in Irvine, CA. The complete pro-
gram and audio files of most presentations are available
on the NAS Web site at www.nasonline.org/telomerase_
and_retrotransposons.
Author contributions: Q.Z., N.-K.K., and J.F. wrote the
paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. N.F.L. is a guest
editor invited by the Editorial Board.
1
To whom correspondence should be addressed. E-mail:
feigon@mbi.ucla.edu.

www.pnas.org/cgi/doi/10.1073/pnas.1100279108 PNAS | December 20, 2011 | vol. 108 | no. 51 | 20325–20332

CR4/CR5 domain (nucleotides ∼243 to
∼326), and the remaining motifs comprise
the core domain, also known as the tem-
plate/pseudoknot domain or the pseu-
doknot/core domain (nucleotide 33–191)
(46, 47, 63). These two highly conserved
structural domains independently bind
TERT (50, 64) and are the only required
hTR elements for in vitro reconstitution
of catalytically active telomerase with
hTERT (49, 50). The 3′ end of hTR has
been identified as an H/ACA small Cajal
body (CB) -specific RNA (scaRNA; nu-
cleotides ∼211 to ∼237 and ∼334 to 451,
the upper boundary of the 5′ hairpin has
not been determined) (59, 62, 65) (Fig. 1),
and it plays essential roles in biogenesis
and regulation of telomerase holoenzyme
in vivo, including accumulation, 3′-end
processing, and localization of hTR (58–
60, 66). The hTR scaRNA binds two sets
of the four evolutionary conserved H/ACA
RNP proteins (dyskerin, Gar1, Nop10, and
Nhp2) to form an H/ACA RNP (Fig. 1)
(67). Structures of single hairpin H/ACA
RNPs from archaea have provided insight
into the likely placement of proteins on
each hairpin of the hTR scaRNA domain
(66, 68–71). The hTR scaRNA also con-
tains a conserved Cajal body localization
element (CAB box) (59) in its 3′ terminal
hairpin loop in the region identified as the
CR7 domain, identifying it as a scaRNP.
The protein telomerase Cajal body protein
1 (TCAB1)/WD-repeat domain 79
(WDR79) that binds the CAB box and
drives localization of hTR into Cajal body
was recently identified (72, 73).
To date, there are no crystal structures of
telomerase RNA or telomerase protein–
RNA complexes; however, over the last
several years, structures of several key
hTR elements have been determined by
NMR spectroscopy (52, 54, 55, 60, 74–76).
This review summarizes what NMR
structural and dynamics studies, combined
with biochemical and mutational analysis,
have revealed about the functional roles of
hTR domains and the overall architecture
of hTR.
Core Domain of hTR
The core domain has been the main focus
of biochemical and biophysical studies of
hTR, because it contains most of the
conserved nucleotides and most of the
disease-linked mutations (21–24, 26, 48,
62). It is the largest functional RNA do-
main at the 5′ end of hTR. Biochemical
characterizations have identified nucleo-
tides between residues 33 and 191 as the
minimal hTR core domain required for
telomerase catalytic activity in vitro (63).
This region can be further divided into
three major segments, a large loop con-
taining the template, the P1 helix as the
loop-closing helix, and a full-length P2/P3
pseudoknot (Fig. 2A). Within the P2/P3
20326 | www.pnas.org/cgi/doi/10.1073/pnas.1100279108
Core Domain
TERT
CR4/CR5
CAAUCCCAAUC
TCAB1
NHP2
NOP10
GAR1
Dyskerin
n (13) destabilizes the pseudoknot. Al-
CR7
5’
box H ACA
3’
H/ACA scaRNA
Fig. 1. Secondary structure and known protein
components of the human telomerase holoen-
zyme. The human telomerase RNA (hTR) contains
three major structural and functional domains,
the core domain, the CR4/CR5 domain, and the H/
ACA scaRNA domain (46, 59, 62). The hTR core and
CR4/CR5 domains independently bind the hTERT
(blue ellipse) (50, 64). The hTR scaRNA domain
binds two sets of the four H/ACA RNP proteins:
dyskerin (green), Gar1 (cyan), Nop10 (magenta),
and Nhp2 (orange) (67). The protein TCAB1/
WDR79 (purple) binds both the dyskerin and the
CAB box located at the CR7 region within the H/
ACA scaRNA domain (72, 73).
pseudoknot, all four helices (P2a.1, P2a,
P2b, and P3) have been shown to be re-
quired for telomerase activity, and disease-
linked mutations disrupting these helical
structures severely impair telomerase ca-
talysis (63). Between these helical regions
are internal loop regions. Except for loop
J2b/3 and the 3′ portion of loop J2a/3,
which are critical for the formation of P2b-
P3 pseudoknot, most of these loop regions
are not conserved, and their nucleotide
identities are not essential for telomerase
activity. The P2b-P3 pseudoknot is linked
to the P1 helix through three nucleotides
at its 3′ end and to the P2a helix through
an asymmetric bulge J2a/b at its 5′ end
(Fig. 2A). On the other side of the P2a
helix are P2a.1 and the adjacent internal
loop J2a.1, which are a mammalian-spe-
cific extension to P2a helix (Fig. 2A). The
5′ end of the P2a.1 helix and 3′ end of the
P1 helix are linked by the large template-
containing loop. These secondary struc-
ture features suggest a unique and overall
compact architecture adopted by the hTR
core domain, because the ends of the ∼43-
bp P2/P3 pseudoknot are constrained
by the intervening 24-nt template-
containing loop.
Structure and Dynamics of the P2b-P3 Pseu-
doknot. The P2b-P3 pseudoknot contains
almost all of the highly conserved nucleo-
tides within the hTR core domain (46).
Early studies of minimal pseudoknots
containing these conserved nucleotides
showed that the pseudoknot unfolds to
a P2b hairpin containing an unusual run of
U-U and U-C base pair (77) and that
a two-base mutation in the P3 found in
some patients with dyskeratosis congenita
though a conformational switch between
the completely folded pseudoknot and this
partially folded hairpin was proposed as
a molecular switch for template trans-
location (77, 78), both the solution struc-
ture of the minimal pseudoknot (discussed
below) and mutational analysis suggest
that a completely folded pseudoknot is
required for optimal activity of telomerase
(52, 79). The solution structure of the
minimal hTR pseudoknot is a compact H-
type pseudoknot with extensive tertiary
interactions between the loop and stem
nucleotides, and it revealed an essential
triple helix in the pseudoknot (52). The
functional importance of the tertiary in-
teractions, described below, was shown by
comparison of thermodynamic stabilities
of pseudoknots with mutations and com-
pensatory mutations and the effects of
the same nucleotide substitutions in-
corporated into the full-length TR on te-
lomerase activity in vitro (52). The tertiary
structure of the minimal hTR pseudoknot
also provided a structural explanation for
the effect of disease mutations in this re-
gion of hTR (52, 54). Mutational studies
and modeling of yeast telomerase pseu-
doknot domains (53, 80) have provided
additional evidence for an essential role
for a conserved triple helix in the pseu-
doknot domain. The putative pseudoknots
in ciliates are much smaller than those
pseudoknots in yeasts and vertebrates.
Although no structures of ciliate pseu-
doknots have yet been reported, sequence
analysis and modeling of 28 ciliate TRs
have predicted one (and in one case, two)
potential base triples in the ciliate pseu-
doknots (81).
In the minimal hTR pseudoknot
(PKDU), the bulge U177 was removed to
stabilize the pseudoknot formation. How-
ever, deletion of this residue in hTR
resulted in a 2- to 10-fold decrease in
telomerase activity (52, 78), and compu-
tational modeling of the P2b-P3 pseu-
doknot suggested that deletion of U177
alters tertiary interactions within the
pseudoknot (82). In addition, substitution
of the 2′-OH of residue A176, which is
adjacent to residue U177, with 2′-OMe or
Zhang et al.
 A 60 G
A A
UCC
U
C
C
G
G
G
A
C
B C D
A G C

P2a.1
CC G C
G G U G
U A U 5’
U C G U
C
G
G
C C 5’
C G

J2a.1
G A
A C C
C U
U G U
A C

J2a/3
U160 C
U C G
G C
P2a U
Template

A U A
C A U
A U G
C
J2a/b

A U A
CG G
C U
CCC
G C
A G C A
C G A U177
A G C
P2b

G G A
U G120C C
C G
G C A
C A U A
C GA
U U A
U
U A
100 U 5’
G U AU177
U
C G
U U
A U
J2b/3

G C P3
U
U U A
C
C G U
U CG C 5’
U
40U U G
U A CU
3’ 3’ 3’
C G 3’
C G
G C P1
G C
5’ 3’
P2b-P3 PKDU P2b-P3 PKWT P2a-J2a/b-P2b P2a-J2a.1-P2a.1

Fig. 2. Structures of subdomains of the hTR core domain. (A) Sequence and secondary structure of the hTR core domain, which can be divided into four
subdomains: P2a-J2a.1-P2a.1 (blue-gray-gold), P2a-J2a/b-P2b (gold-green-red), P2b-P3 pseudoknot (red-pink), and P1 (dark green) (46). (B) NMR solution
structures of the P2b-P3 pseudoknot, where PKDU (PDB ID 2K96) and PKWT (PDB ID 2K96) are structures for ΔU177 mutant and WT constructs, respectively
(54). (C) NMR solution structure of P2a-J2a/b-P2b (PDB ID 2L3E) (55). (D) Structural model of the P2a-J2a.1-P2a.1 determined by the RDC-MC-Sym approach (55).
All 3D structures are color-coded like the secondary structure in A, and nonnative residues are colored light gray.

2′-H results in about a twofold decrease in
activity, leading to the proposal that the
A176 2′-OH may contribute directly to
telomerase catalysis (53). To provide
structural insight into the WT P2b-P3
pseudoknot (PKWT) and the functional
role of U177, the solution structure of
PKWT was determined, the original
structure of PKDU was further refined
with an extensive set of NMR residual
dipolar couplings (RDCs) (Fig. 2B), and
systematic structural and dynamical com-
parisons were made between PKWT and
PKDU (54). These structural character-
izations revealed that PKWT folds into the
same H-type pseudoknot conformation as
PKDU with almost identical tertiary in-
teractions (Figs. 2B and 3 A and B). In
both PKWT and PKDU, the P2a and P2b
helices are stacked on either side of
A A171
U97
A117
G98
C116 U100
U115
A174
U114
A175
U113A176
C112
G178
U177
Fig. 3. Tertiary interactions in the pseudoknot. (A) The structure of the triple helical region of PKWT
(54). (B) Schematic representations of the tertiary interactions in PKWT. In PKDU, U103 forms a Hoogs-
teen base pair with G178. (C) U·A-U Hoogsteen base triple. (D) Detailed view of the base pairs sur-
rounding U177. The base of U177 stacks over the 2′OH of A176. The nucleotides are in CPK colors, except
that U177 is colored magenta.
a junction that is a Hoogsteen base pair helical interactions. Based on the struc-
formed between the first nucleotide (U99) ture, the base of U177 sits right over the
in the J2b/3 loop and the last nucleotide 2′-OH of residue A176 and would steri-
(A173) in the J2a/3 loop, and both pseu- cally block its accessibility during telo-
doknots are stabilized by the same base merase catalysis (Fig. 3D). Dynamic
triples flanking this junction base pair characterization by NMR spin relaxation
(Figs. 2B and 3B). The A-rich J2a/3 loop measurements revealed that U177 is in-
forms two minor groove base triples with trinsically highly flexible, which suggests
the P2b helix, and the U-rich J2b/3 loop that, rather than blocking A176, U177 may
forms three major groove U-A·U Hoogs- serve as a hinge to provide additional
teen base triples with P3 helix (Fig. 3C). backbone flexibility for residue A176 to
However, PKDU is also stabilized by an facilitate the catalysis (54).
additional C112·G178-U103 triple that is The structure determination of PKWT
disrupted in PKWT because of the pres- also revealed why it is thermodynamically
ence of U177, which creates a large roll less stable than PKDU (52). Comparison
and tilt between the flanking base pairs of NMR imino proton spectra of PKWT
(Fig. 3 A and D). In PKWT, U177 is flip- and PKDU as a function of temperature
ped out of the P3 helix into the minor revealed an unanticipated difference in
groove and is located on the opposite side the late folding/early unfolding pathway.
of the helix from the major groove triple In PKDU, as expected, when temperature
increases, the Hoogsteen base pairs be-
tween the J2b/3 loop and P3 helix begin to
B C melt before the Watson–Crick base pairs
3’ 5’ 5’ in P3 helix. However, in PKWT, the
G118 C96 C170
U
A172 A117 U97
presence of bulge U177 results in partial
A171
U unstacking of the three Watson–Crick U-
C116 G98 A172
A173 A base pairs from the rest of the helix
A173 U99 A below the bulge U177 (Fig. 3A). As a
U115 A174 U100 consequence, the three U-A base pairs
U114 A175 U101 D above and the two base pairs below the
U101 U113 A176 U102 A176 bulge U are thermodynamically less stable
U113
U102 U17
7 U1 than the tertiary (Hoogsteen base pair)
C112 G178 03 C112
interactions, and they begin melting before
A111 U179
C104 the Hoogsteen U-A base pairs. Thus, the
U103 G110 C180 G178
5’ presence of U177 in PKWT results in an
3’ 3’
altered late folding/early unfolding path-
way in the pseudoknot, which may be rel-
evant to RNA assembly. In addition, for
both pseudoknots, the melting studies re-
vealed that the junction J2b/3 loop–J2a/3
loop Hoogsteen U-A base pair is

Zhang et al. PNAS | December 20, 2011 | vol. 108 | no. 51 | 20327
remarkable stable. Thus, this base pair
plays a critical role in overall folding of the
pseudoknot (54).
Structure, Dynamics, and Function of the P2a-
J2a/b-P2b. Adjacent to the highly conserved
P2b-P3 pseudoknot is a 5-nt bulge loop,
J2a/b, which serves as a bridge between the
pseudoknot and the P2a helix. This py-
rimidine-rich J2a/b bulge loop is not highly
conserved in sequence, except for a rela-
tively conserved G at the 5′ end (61% in
vertebrate and 83% in mammalian TRs)
(39). Initial studies showed that swapping
the bulge sequence 5′→3′ or replacing the
hTR sequence with the mouse TR se-
quence had little effect on human telo-
merase catalytic activity (63, 83). These
results, along with the lack of sequence
conservation, argued against an important
role of the J2a/b bulge loop in telomerase
activity. However, the location of the
J2a/b bulge loop is conserved in all verte-
brate TRs, and its length is usually 5 nt in
mammalian TRs (46, 47). The solution
structure of the J2a/b bulge loop together
with flanking P2a and P2b helices showed
that J2a/b introduces a large bend (89 ±
3°) between P2a and P2b across the major
groove (Fig. 2C) (55). The 5′-end residue
of the bulge loop, G84, stacks above P2a,
and the 3′-end residue of the bulge loop,
C88, stacks below P2b. A change in the
backbone direction occurs at the center
bulge residue, U86, which leads to an
overall S-shape conformation of J2a/b.
Remarkably, the S-shape structure of the
J2a/b results in almost no twist between
P2a and P2b (−10 ± 10°) (Fig. 2C). A
search for other 5-nt bulge structures, us-
ing the FRABASE program (84), un-
covered only one other 5-nt bulge among
all RNA structures solved to date from the
hepatitis C virus (HCV) internal ribosome
entry site (IRES) domain II (85). Even
more surprisingly, this HCV IRES domain
II bulge adopts the same S-shaped struc-
ture, despite the fact that it has a different
sequence from the hTR J2a/b. A some-
what broader search that also allowed for
noncanonical closing base pairs and
swapping the strand with the 5-nt bulge
uncovered two additional sequences, and
these sequences have similar S-shaped in-
ternal loops (86, 87). Thus, the J2a/b bulge
represents a rare structural motif.
Systematic dynamic characterizations by
NMR 15N spin relaxation (88) and NMR
RDCs (89, 90) also revealed another un-
usual feature of the J2a/b bulge loop. It
was initially expected that the J2a/b bulge
loop would be highly flexible, but although
J2a/b exhibits some flexibility, its motion is
remarkably limited on the nanosecond to
millisecond time scale compared with the
3-nt bulge loop in HIV-1 transactivation
response element (TAR) RNA. Based on
a cone motional model, P2a and P2b move
20328 | www.pnas.org/cgi/doi/10.1073/pnas.1100279108
relative to each other with a motional
amplitude of ∼39°. In contrast, HIV-1
TAR RNA has a cone-model motional
amplitude of ∼55° (91).
The rare structure and unexpected dy-
namics of J2a/b suggested that it might
have an important functional role in telo-
merase RNA topology and function. To
test this hypothesis, systematic mutations
were carried out to investigate the effects
of the length, strand location, and sequence
of J2a/b on telomerase function (55). The
results showed not only that the di-
rectional bending defined by J2a/b is re-
quired for overall telomerase activity but
also that the intrinsic flexibility across J2a/
b is important for processive catalysis by
telomerase. The results also suggest the
consensus sequence G83Y78Y87Y96Y87 for
the 5-nt bulge in mammalian TRs may
have evolved for both nucleotide addition
and template translocation during telo-
merase catalysis. Finally, the large bend at
the J2a/b bulge would be expected to play
a significant role in determining the over-
all topology of the core domain, which
discussed more below.
Structure and Dynamics of the Mammalian-
Specific P2a.1-J2a.1-P2a. Located on the
other side of hTR P2/P3 pseudoknot is the
P2a.1-J2a.1-P2a domain, where P2a.1 is
a mammalian-specific extension to the P2a
helix through an asymmetric internal loop
J2a.1. Biochemical studies have shown that
some nucleotide substitutions in this region
and the C72G mutation associated with
aplastic anemia result in a decrease in
telomerase activity (62, 63, 83). In NMR
spectra of a P2a.1-J2a.1-P2a construct,
most of the nonexchangeable protons
from the J2a.1 loop and surrounding nu-
cleotides were exchange-broadened and in
many cases, not visible. This finding not
only indicated the presence of significant
conformational exchange in J2a.1, appar-
ently because of the bases in the internal
loop adopting more than one conforma-
tion involving alternative base pairs, but
also hindered high-resolution structure
determination of this region of hTR by
solution NMR (55). To overcome these
difficulties, the RDC-MC-Sym approach
was developed for structure determination
of nucleic acids (55). This approach uses
a combination of computational modeling
by program MC-Sym (92) and experi-
mentally derived restraints by NMR
RDCs. The structural analysis revealed
that P2a.1 is essentially a linear extension
of the P2a helix (Fig. 2D). The interhelical
bend between P2a.1 and P2a is only 6 ± 3°,
and the twist between the two helices is
135 ± 14°, which agrees well with the
amount of twist for an ∼4-bp irregular
helix formed by the asymmetric loop J2a.1.
Although J2a.1 undergoes significant
conformational exchange, the interhelical
motion between P2a.1 and P2a is re-
markably restricted (∼26° cone motions)
based on dynamic characterization by
NMR RDCs. The stability between
helices is consistent with the observations
that disruption of base pairs flanking
J2a.1 decreases telomerase activity and
that nonmammalian vertebrate TRs
have a single long P2a helix without a
J2a.1 (46, 47).
High-Resolution Model of the hTR Core Do-
main. The structures and dynamic anal-
ysis of subdomains of the hTR core do-
main, in particular, the conserved
pseudoknot, have provided great insights
into their functional roles in telomerase
catalysis. However, they do not provide an
overview of how the P2/P3 pseudoknot
folds and how its architecture imposes
conformational constraints that position
the template into the active site of telo-
merase. A low-resolution (6.5–8.0 Å)
FRET model of the hTR core domain has
been reported (PDB ID 2INA), where
distances derived from FRET between
fluorescently labeled peptide nucleic acids
were used in structural modeling (93). In
this approach, although these distances
were obtained in the full-length hTR, they
provide only indirect constraints on the
positions of the helical regions of the hTR
core domain, because the peptide nucleic
acids were hybridized onto three single-
stranded regions, the 5′ end of hTR (nu-
cleotides 1–13), the template region (nu-
cleotides 44–56), and the J2a/3 region
(nucleotides 146–158). One caveat is that
the inherent flexibility of the single-strand
regions, in particular, at the 5′ end of the
hTR, was not considered in the modeling.
Recently, by applying a computational
modeling approach that incorporates the
Assisted Model Building with Energy Re-
finement (AMBER) force field and short-
range NOE restraints from individual
structures, a high-resolution structure
model of the P2/P3 pseudoknot was de-
termined that contains all nucleotides ex-
cept for the nonconserved single-strand
region of J2a/3 (Fig. 4A) (55). There are
significant differences between the FRET-
and NMR-based structure models in both
the local structures and relative helical
orientations, but both models do exhibit
an open architecture of the hTR core do-
main and a major interhelical bend oc-
curring across the J2a/b bulge (55, 93).
The NMR-based structure model
revealed that the full-length P2/P3 pseu-
doknot of the hTR core domain folds into
an overall V-shape conformation that is
defined by the ∼90° bend across the J2a/
b bulge. The P2b-P3 pseudoknot and
P2a.1-J2a.1-P2a domain are located on
either side of the J2a/b bulge, and they are
each ∼50Å long. Thus, the bend across
J2a/b creates an ∼70 Å end to end
Zhang et al.
 A B
Palm Fingers

A176
U177
DNA

Thumb TRBD
A62
Template TEN CP T 1 2 A B’ C D E CTE
Human TERT
Tribolium castaneum TERT
3’ 5’

Fig. 4. Models of the hTR core domain and interaction with TERT. (A) NMR-based model of the hTR core domain including a DNA primer bound to the
template (55). (B) The hTR P2/P3 pseudoknot positioned onto the T. castaneum TERT in two possible orientations, where the hTR P2/P3 pseudoknot lies either
parallel (Left) or perpendicular (Right) to the T. castaneum TERT-telomeric RNA/DNA complex (PDB ID 3KYL) (37). The color scheme for the hTR P2/P3 pseu-
doknot domain is the same as in Fig. 2. Domains of the T. castaneum TERT are colored and labeled as shown, and the RNA template and telomeric DNA are
colored in purple and cyan, respectively. A comparison of the domain structures of the hTERT and T. castaneum TERT is also shown.

distance between P2b-P3 and P2a.1, which
agrees well with the length of the in-
tervening 24-nt single strand containing
the template without significant stretching.
Because of the negligible twist between
the P2a and P2b induced by the J2a/
b bulge loop, the structural model also
revealed that the conserved bulge residue
U177 (52, 78) and the catalytically im-
portant 2′OH of residue A176 (53) are
both positioned on the inner surface of the
core domain and face to the 5′ end of the
template, where the active site should be
located. The structural model is also con-
sistent with the close proximity of the
end of the pseudoknot to the template
needed to obtain maximal activity in an
engineered cis-telomerase (94). In addi-
tion, the dynamic characterization showed
that interdomain motion between P2a and
P2b has an amplitude of ∼39° assuming
a cone motional model, which can be
translated into a displacement of ∼28 Å
between the two ends in the full-length P2/
P3 pseudoknot. This distance agrees well
with the ∼17 Å that the template must
translocate through the active site during
the synthesis of each TTAGGG telomere
repeat. Although the binding sites of
TERT on the core domain have not been
identified, the overall shape of the P2/P3
pseudoknot fits nicely into the crystal
structure of the TERT from T. castaneum
(36, 37). The pseudoknot can be modeled
onto the TERT either in parallel or per-
pendicular to where the template/primer
fits in the donut hole of the TERT, such
that flexing of J2a/b would allow the tem-
plate to be pulled through the active site
during telomere synthesis (Fig. 4B). This
finding provides a testable model for how
the core domain interacts with TERT.
STE of hTR
The STE is the other catalytically essential
TR element conserved across ciliates,
vertebrates, and yeasts (9, 50, 95, 96). In
hTR, the CR4/CR5 domain is the STE,
and it interacts directly with hTERT in-
dependently from the core domain. The
hTR CR4/CR5 domain contains a three-
way junction (Fig. 5A), which has also
been proposed to form in yeast telomerase
RNAs (97). Like the core domain, al-
though the CR4/CR5 secondary structure
is conserved, most conserved residues are
localized in one particular region, the P6.1
hairpin. Additional highly conserved resi-
dues are located at the large internal loop
that forms a three-way junction between
P6, P6.1, and P5. Although various Wat-
son–Crick base pairs can be drawn be-
tween nucleotides in the three-way
junction, substitutions and compensatory
mutations indicate that it is the sequence
rather than the base pairs that are impor-
tant for telomerase activity (98). In addi-
tion, a minimal CR4/CR5 domain
construct, which includes P6.1, all of P6a-
P6b except the terminal hairpin, and
flanking nucleotides but does not include
the P5 helix, is sufficient to reconstitute
telomerase activity in trans with the core
domain and TERT (Fig. 5A) (76). Thus, it
is not clear whether this region forms
a three-way junction in the context of its
association with TERT. To date, no
structural information on the CR4/CR5
three-way junction has been reported, but
the structures of two helical regions of the
hTR STE have been solved (74–76). These
structures are the essential P6.1 hairpin
and the central portion of P6 surrounding
the J6 internal loop.
Structures of P6.1 and Pseudouridylated P6.1
The P6.1 is a short hairpin off of the three-
way junction that has a helix of four
Watson–Crick base pairs capped by a 5-nt
loop. The 13-nt sequence of P6.1 is highly
conserved; in addition to U307 and G309
in the loop, all 8 nt forming Watson–Crick
base pairs are 100% conserved in verte-
brates. The formation of base pairs in P6.1
has been shown to be essential for TERT
binding and telomerase activity (50, 98).
Both the length of the P6.1 helix and the
two conserved loop residues are critical for
telomerase activity but not for binding of
TR to TERT (64, 98). The structure of
P6.1 showed that the first (U306) and the
last (G310) residues of the 5-nt loop form
a U-G wobble pair on top of the A-form
helix (Fig. 5B) (75). The remaining three
residues from the loop, U307, G309, and
G310, are exposed to solvent, with U307
and G309 located on the minor groove
side of the loop and G308 located on the
major groove with partial stacking on
U306. The small loop formed by these
three residues has a well-defined confor-
mation, and this architecture of the loop
has been proposed to allow the essential
loop nucleotides to interact with TERT or
TR (75). Intriguingly, gel shift and cross-
linking experiments have shown that iso-
lated hTR fragments of P6.1 and the
template can directly interact with con-
tacts between the P6.1 loop residues and
the two ends of the template region (99),
although the biological relevance of this
interaction in the context of hTERT re-
mains to be established.
Most noncoding RNAs contain modified
nucleotides, and hTR seems to be no ex-
ception. Six potential pseudouridine (Ψ)
modification sites have been identified
within the hTR, two of which are located
in the P6.1 loop (76). Pseudouridine is the
most abundant posttranscriptional modi-
fied RNA nucleotide, and it is found in all
species (100). The locations of pseudour-
idines are usually highly conserved, and
they play important roles in biological
functions. To investigate the potential
functional roles of these Ψ-modifications
in the essential P6.1 loop, the solution
structure of a pseudouridine-modified
P6.1 (Ψ-P6.1) was determined (Fig. 5B)
(76). Furthermore, both the thermody-
namic stability of the Ψ-P6.1 vs.

Zhang et al. PNAS | December 20, 2011 | vol. 108 | no. 51 | 20329
A U
C C
G B P6.1 Ψ4P6.1 pseudouridylation may have a subtle but ugAG sequence and serves as a CB local-
U307 Ψ307
C G
U A significant effect on telomerase activity. ization signal (105). Remarkably, the CR7
U G
C G U306 Ψ306 domain of vertebrate TRs, as discussed
P6b G C
A Structure of the P6 Hairpin. The P6 hairpin is below, contains not only a CB localization
270 G C Minimal
G C
G C CR4/CR5
located adjacent to the 5′ end of P6.1. It signal but also another signal for the ac-
J6 C
C
A
C 290
5’
5’
has two helical regions, P6a and P6b, that cumulation and processing of TRs (60).
flank a small asymmetric internal loop J6,
U
C G
P6a G C Although the H/ACA and CR7 domains
G C
P6 and it is capped by a UCCG hairpin (Fig. are not required for telomerase activity
U
C A
260 G C
C U
U C
G 5A). Deletion of the hairpin loop has no
G C
U
U G C G in vitro, they form a scaRNA domain
C G
G C A
G G
G 310
A U P6b
G C
effect on telomerase activity, but addi- that is essential for in vivo accumulation,
C GA G C A
G
C A
C
U
P6.1
C
C U
C 290 tional deletions that include the J6 in- 3′-end processing, and localization of
U C G
A
G
G G C ternal loop have been shown to inhibit
G U G C vertebrate TRs.
G 250 C C interactions between CR4/CR5 and TERT
U G
A U A
P6a Although no high-resolution structures
C
C GC
C 260 G C
and abolish telomerase activity (50).
G C
G C
A U of the H/ACA domain from any verte-
P5 C G G C The solution structure of an RNA de-
C G 5’ 3’ brate TRs with or without H/ACA RNP
C G 5’ rived from P6, including the J6 internal
5’ 3’ proteins have been determined to date,
loop, has been determined (Fig. 5C) (74).
Fig. 5. Structures of subdomains of the hTR CR4/ P6a was predicted on the basis of phylo- a combined structural and biochemical
CR5 domain. (A) Sequence and secondary struc- genetic analysis to have a C262-A295 base study, including localization studies using
ture of the hTR CR4/CR5 domain (46). The minimal pair next to bulge U261, but in the struc- FISH, on the hTR has delineated the
CR4/CR5 required for reconstitution in vitro of ture, a U261-A295 base pair forms and
active telomerase is highlighted by a dashed box
C262 is bulged out. The J6 internal loop
(76). (B) NMR solution structures of P6.1 (PDB ID
1OQ0) (75) and Ψ4P6.1 (PDB ID 2KYE) (76). The
is well-defined, with a potential triple A CR7 B g414
g C C415
formed between C267 through water-

CAB Box
two U and three G letters in the P6.1 loop are A U A413
colored in green and yellow, respectively. The two mediated hydrogen bonds to the G268- g412
U416
C288 base pair. Interestingly, similar to the g g g417
pseudouridines (Ψs) in the P6.1 loop are colored in
red. (C) NMR solution structure and secondary P2a.1-J2a.1-P2a, the interhelical motion u U u411
U418
structure of a P6 hairpin construct (PDB ID 1Z31) 410 C G
between P6a and P6b across this asym- C410
G419
(74). The internal loop residues (C266-C267 and metric bulge is very limited with amplitude C G 420 C409 G420
A289-U291) are colored in green, and the bulge G C
<10°, which was revealed by RDC analysis.
residue C262 is colored in purple. U A 3’ 5’
These structural and dynamical properties 5’ 3’

of J6 result in a unique conformation of

C D

G414
unmodified P6.1 and the effect of pseu- the P6 region. In addition to a 20° inter- C415
helical angle and ∼3 Å deflection between
douridylation on telomerase activity in vi- A413 U416
P6a and P6b, the J6 internal loop forms an
tro were characterized. The loop structure G412
G41
7
unusual solvent-accessible opening, lead-
of the Ψ-P6.1 is significantly different from ing Leeper et al. (74) to speculate that this
U41
1
U418
C410 G419
the loop structure of P6.1. The stem clos- internal loop may be important for TERT C409 G420
ing Ψ306·G310 base pair has a single hy- or TR interactions. However, a variety of C408 G421

drogen bond between the imino proton of base substitutions in this internal loop do 5’
U407 A422
3’
G310 and the O4 of Ψ306, which is dif- not abrogate activity (50), and therefore, it
ferent from the canonical U·G wobble pair E F
G414

is not clear at the current stage that this

found in the unmodified P6.1. The bases structure is unique. C415

of Ψ306 and Ψ307 are located in the major A413 U416

groove of the loop and are stacked on H/ACA scaRNA Domain of hTR G412
G41
7
1
U41
G305 and Ψ306, respectively. These two In vivo biogenesis and regulation of telo- U418
C410 G419
pseudouridines are spatially positioned to merase holoenzyme require additional C409 G420
form water-mediated hydrogen bonds be- telomerase RNA motifs. In vertebrates, the C408 G421

tween the imino protons and their 5′
phosphate oxygens. Only one base, the
nonconserved G308, is flipped out of the
loop, and it is on the minor groove side.
The phosphate backbone turns between
G308 and G309, and G309 in the syn
conformation stacks on the sugar of G310.
These structural features revealed that the
Ψ-modifications at the loop increase both
base-stacking and hydrogen-bonding in-
teractions in the Ψ-P6.1 relative to the
unmodified P6.1, resulting in higher ther-
modynamic stability that is characterized
by UV melting studies. Interestingly, the
pseudouridine modification at 306 and 307
in the P6.1 loop decreased the telomerase
activity in vitro by approximately threefold
but slightly increased the telomere addi-
tion processivity (∼20%), indicating that
3′-half of TR comprises an H/ACA scaR-
NA domain, which includes a telomerase-
specific CR7 domain that forms a 3′-ter-
minal hairpin. All H/ACA small nucleolar
RNAs (snoRNAs) and scaRNAs form
RNPs with evolutionary conserved H/
ACA RNP proteins and generally function
to direct site-specific pseudouridylation of
ribosomal RNAs and small nuclear RNAs,
respectively (101–104), but in the case of
telomerase, the domain seems to have
been co-opted to help localize telomerase
to Cajal bodies (56, 58, 59). The H/ACA
motif has a hairpin-hinge-hairpin-tail sec-
ondary structure, where the conserved H
box is located at the hinge region and the
ACA box is located at the 3′-end tail (66).
In addition to the H/ACA motif, scaRNAs
share another common motif, known as
the CAB box, which has a consensus
5’
U407 A422
3’
Fig. 6. Structure and function of the hTR CR7
domain (60). (A) Sequence and secondary struc-
ture of the hTR CR7 hairpin and flanking base
pairs. Boxed residues are the CAB box sequence.
Residues with >95% and >85–95% conservation
among all vertebrate species are shown in capital
letters and bold fonts, respectively. Nonnative
residues used in structure determination are the
base pairs below the dashed line. (B) NMR solution
structure of the CR7 hairpin (PDB ID 2QH2). Resi-
dues are colored by type: orange, A; green, U;
blue, G; red, C. (C and D) Residues comprising the
hTR-specific processing signal are highlighted in
magenta in the secondary structure (C) and col-
ored as in B in the surface representation of the
CR7 structure (D). (E and F) Residues comprising
the CB localization signal are highlighted in dark
blue (CAB box) and light blue in the secondary
structure (E) and colored as in B in the surface
representation of the CR7 structure (F).

20330 | www.pnas.org/cgi/doi/10.1073/pnas.1100279108 Zhang et al.

sequence and structural elements of the
two independent signals, located in CR7,
for 3′-end processing and accumulation
and localization of hTR to Cajal bodies
(Fig. 6) (60). The CR7 hairpin loop has
four key structural features. First, within
the 8-nt loop, there is 1 bp observed be-
tween the first and the second to last nu-
cleotides of the loop, a U411·G417 wobble
pair. Second, because of the U·G pair in
the loop and stable Watson–Crick base
pairs in the helix, the last nucleotide of the
loop, U418, adopts an unpaired confor-
mation. Third, the first 3 loop nt that are
also the first 3 nt of the CAB box are
stacked over each other, forming A-form
backbone geometry. Finally, although the
last CAB box residue (G414) does not
stack over other residues and is quite
flexible, a change in the backbone di-
rection occurs between G414 and C415,
positioning all CAB box residues on the 5′
side of the loop. To determine which of
these structural features is important for
the 3′-end processing and localization
signals, parallel structural determinations
were performed on the terminal loop
construct derived from the human U64 H/
ACA snoRNA and human U85 C/D-H/
ACA scaRNA. It had been previously
shown the U64 snoRNA replacement of
the hTR CR7 in the full-length hTR al-
lows hTR accumulation and processing,
but the RNA localizes to nucleoli instead
of Cajal bodies. In contrast, replacement
of the CR7 hairpin with the 3′ hairpin of
U85 scaRNA abrogated processing, and
the hTR remained at the site of tran-
scription. Comparison of the terminal loop
structures of the hTR CR7 and the 5′
hairpin of human U64 H/ACA snoRNA
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and potentially each other to facilitate
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remain to be determined. Combined
use of NMR, X-ray crystallography,
small-angle X-ray scattering, single mol-
ecule techniques, and EM will likely re-
veal these structures within the next
few years.
ACKNOWLEDGMENTS. Q.Z. is a Baltimore Family
Fellow of the Life Sciences Research Foundation.
This work was supported by grants from the Na-
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Foundation (to J.F.).
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